The broad, long term objective of this research program is to define at a molecular level the mechanism(s) and regulation of signal transduction by thyrotropin-releasing hormone (TRH) and its GTP-binding regulatory (G) protein-coupled receptor (TRH-R). These studies will lead to a better understanding of cell regulation under physiologic and pathologic circumstances by TRH and all hormones, growth factors and neurotransmitters that signal via receptors of this family. The findings may lead to rational design of new drugs to treat diseases of these signalling systems. This proposal is timely because new insights into these diseases are now being gained and a combined approach using molecular biology and computer modelling holds great promise for a deeper understanding of these processes. Three interrelated aspects of TRH-R biology will be explored. The Specific Aims are: #1. To model the 3-D structure of TRH-R. 1A. To further define the atomic interactions between TRH and TRH-R. 1B. To further define the intramolecular interactions among transmembrane helices of TRH-R. 1C. To develop a model of the extracellular loops of TRH-R and determine whether these loops serve to direct TRH into the binding pocket. #2. To delineate the mechanism of TRH- R activation. 2A. To identify the G protein coupling domain(s) of TRH-R and determine the specific amino acids involved in receptor/G protein coupling. 2B. To identify transmembrane residues in TRH-R that are important for activation. 2C. To develop a computer simulation of the conformational changes in TRH-R that are associated with transition from the inactive to the activated state. #3. To identify the sequences in TRH- R that mediate the post-translational processes of internalization and downregulation, and to delineate these mechanisms. For Specific Aims #1 and #2, an interactive approach using tools of molecular biology to experimentally analyze structure-function of TRH-R will be combined with computer simulations that are based on the experimental observations and will guide further experimentation. For Specific Aim #3, mutagenesis of TRH-R will be combined with cell biologic studies. Experiments will be performed by gene transfer of native and mutated proteins into cell culture systems.